“Prionins”, highly specific markers for noninvasive presymptomatic detection of TSE diseases, and targets for therapeutic reagents to prevent and control TSE diseases in animals and humans

ABSTRACT

Proteins expressed from within the prion protein genes of all animals and humans, “prionins”, against which reagents can be prepared for accurate pre-symptomatic diagnosis, for detecting latent TSE, for detecting TSE contamination of food, blood and blood products and for therapeutic treatment of Bovine spongiform encephalopathy (BSE) in cows, Scrapie disease in sheep and Creutzfeldt-Jacob syndrome in humans, are revealed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to PCT ApplicationsPCT/EP98/03609 filed Jun. 16, 1998 and CA 2,206,774 filed Jun. 16, 1997.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

This invention is related to diagnostic and therapeutic molecules forthe detection, prevention of bovine spongiform encephalopathy (BSE),scrapie disease (scrapie) and Creutzfeldt-Jakob syndrome (CJS).Specifically, the invention relates to three closely related proteinsBSAS, SCRAPAS and CJAS which are implicated in causing BSE, scrapie andCJS, to antagonists of these proteins, to diagnostic reagents to detectthese proteins in clinical samples and food, and to therapeutic methodsdirected at these proteins in animals and humans. It is also related tothe use of homologues of these proteins from hamsters and mice which areuseful for developing and testing methods for use with vaccines andother agents for therapeutic value in treatment of humans and animalsfor TSE diseases.

BACKGROUND OF THE INVENTION

Prion proteins (PrPs) are a family of very closely related proteinswhich are found in a number of allelic forms in the membrane of certainpopulations of brain neurons of all animals. PrPs are expressed inlymphoreticular system and replicated in spleen and otherlymphoreticular tissues (Kimberin R. H. and Walker C. A. Virus Res.12:201-211 (1989). The alteration of the molecular configuration(folding) of a PrP by an unknown mechanism which converts these proteinsinto infectious particles “prions” (PrPsc) is associated with a group ofdiseases called “Transmissible spongiform encephalopathies” (TSE)(Prusiner S. B. Science 252:1515-1522 (1991); Prusiner S. B. Ed.,Current topics in Microbiology and Immunology 207 (1996)); (Westaway Det al., Trends. Microbiol. 3:141-147 (1995)); (Goldfarb L. G. and BrownP. Annu. Rev: Med. 46:57-65 (1965)).

Three members of the TSE family are of great economic and medicalconcern; these include Bovine spongiform encephalopathy (BSE) in cows,scrapie in sheep and Creutzfeldt-Jakob syndrome (CJS) in humans.Pathology of TSE involves nerve cell dysfunction, which leads to fatalneurodegeneration; TSEs are characterised by symmetrical vacuolation ofneurons and neuropil and accumulation of PrPSc around neurons. Thelatter phenomenon is believed to be the cause of the phenotype of thedisease in the affected animal or human (Darcel C. Vet. Res Commun.19:231-252 (1995)).

Prions are believed to be self-replicating proteins, which in theiraltered configuration are resistant to destruction by proteolyticenzymes and heating conditions which usually, destroy most proteins. Theinfectious prion is believed to be transmissible across species.Nevertheless, it has not been satisfactorily explained how a brainresident protein which, so far has not been demonstrated in biologicalfluids, can be transmitted from animal to animal within a grazing herdof cattle. Also, it is a puzzle that an endogenous protein auto-convertsfrom a harmless, useful, form to a highly pathogenic infective form;many exotic hypotheses have been proposed to explain these phenomena.

However a few plausible hypotheses have provided insight into someaspects of the above mentioned puzzle, e.g., (i) it has been shown thatPrPsc can be detected in the tonsils of animals with BSE and scrapie andpresumably, animals presymptomatic for the above diseases (Schreuder B.E et al., Nature 381:563 (1996); (ii) it has been suggested that prionscould be receptors that ushers an unknown virus or other infectiousagents into cells (Brown P. (quoted in special news report) “PuttingPrions to the Test” Science 273:184-189 1996)); (iii) experiments inmice appear to indicate that PrP binds to a chaperon protein (protein X)which catalyses the formation of PrPSc (Telling G. C. Cell 83:79-90(1995)); (iv) that an unidentified factor is responsible for BSE andprions act as host-adapting agent for the factor (Lasmezas C. I. et alScience 275:402-405 (1997), and (v) that a frame shift translation inscrapie PrP mRNA may play a role in conversion of PrP to PrPsc, (WillsP. R. et al. Microbiol. pathogenesis 6:235-249 (1989); Kiyotoshi K. etal.; Proc. natl. Acad. Sci. USA 94:10069-10074 (1998).

There is an intriguing relationship between Scrapie in sheep, BSE incows and CJS in humans; it is believed that BSE is initiated by thescrapie prion and some forms of CJD are initiated by eating BSE infectedcows, particularly brain. The latter appeared to be experimentallysupported by the discovery, recently, of what has been called a BSEsignature molecule in humans who contract a “new variant of CJD(Collinge, J et al., Nature 283:685-690 1996. Nevertheless despite alarge international effort to understand the pathogenesis of TSE, and todevelop non invasive widely applicable methods for presymptomaticdetection of these diseases, which in the case of BSE and scrapie havecalamitous effects on commercial activity and on the health of thepopulation at large, substantial progress has not been made in thesedirections.

SUMMARY OF THE INVENTION

The object of the present invention was to find other proteins whichwere expressed in a disease-specific manner, and which might interactwith and convert PrP to PrPsc. We reasoned that such proteins whichwould occur in body fluids, would be highly specific ante-mortemdiagnostic markers in pre symptomatic cows, sheep and humans and theywould be useful as therapeutic targets to manage the symptoms of TSEdiseases in animals and humans. Furthermore, we aimed to invent amethod/methods, which would detect cross infection of humans and otheranimals by such agents that cause BSE and scrapie.

According to the invention the solution to the problem was to use theimplied relationship between human Alzheimer's disease (AD) and theprion diseases, especially the relationship of a protein ALZAS, which wehave discovered to be the potential causative factor of all forms of AD.ALZAS (Bergmann J. E. et al., Neurobiology of Aging 17:S14 (1996), aprotein encoded and expressed within the human APP gene; the latter isstrongly linked to Alzheimer's disease (AD). The Post-translationalmodification of the APP gene product the “β amyloid precursor protein”is the β amyloid protein (Aβ). Aβ plays a central role in thepathophysiology of AD (Scheuner D. et al., Nature Medicine 2:864-870(1996)). Recently it was shown that protein products of frame shiftmutations in the APP gene and the ubiquitin β gene might be involved inthe pathology of Alzheimer's disease and Down syndrome (van Leeuwen F.et al., Science 279:242-247, (1998). ALZAS which is made up of Aβ, theAPP protein transmembrane signal sequence and a unique intron encodedc-terminal sequence, is detected in brain, blood and saliva of allhumans with Alzheimer's disease (AD), and has the predicted biochemicalcharacteristics to initiate the clinical symptoms of AD (Bergmann J. E.et al., Neurobiology of Aging 17:S14 (1996)). The pathophysiologicalsimilarities between AD and the TSEs, particularly CJD, and the impliedrelationship of ALZAS to APP/Aβ led us to search for a molecule similarto ALZAS within the prion protein genes of cattle, sheep and humans, andin mice and hamsters. The two rodent species which have been usedextensively by scientist wanting to model TSE diseases. Using the methodwhich we call disease “gene discovery by positional searching” (DGDPS),(see Bergmann J. and Preddie E. “WIPO” publications No# WO98/07850 andNo#WO98/07851). Our search led to the discovery of proteins BSAS,SCRAPAS and CJAS, which are encoded and expressed from within the PrPgenes of cattle, sheep and humans respectively. Additionally wediscovered MOPAS and HAMPAS, which were encoded within the PrP genes ofmice and hamsters. Since these proteins were discovered within thechromosomal region encoding prion protein genes we named them“prionins”. Following is a brief description of DGDPS as it was appliedto the discovery of prionins

Relationship of BSAS and SCRAPAS to BSE and Scrapie

These proteins appear to be specifically related to the development ofBSE in cattle and scrapie in sheep. BSAS and SCRAPAS appear in the bloodof all animals with clinical symptoms of the disease and in asignificant percentage of animals that have been exposed to the disease,but not usually in animals that have not been exposed to the disease. Inaddition, animals exposed to the disease produce specific antibody(endogenous antibody) directed against BSAS or SCRAPAS, which can bedetected presymptomatic in the sera of affected animals. Theconcentration of the endogenous antibody in the serum of animals withclinical symptoms of the disease appears to be generally lower than thatof the animals without clinical symptoms of the disease, which led us tospeculate that there may be a weakening of a subjects immune defence atinitiation of the disease.

BSAS and SCRAPAS

BSAS and SCRAPAS are relatively small β-sheet derived proteins expressedfrom within the prion protein genes of cows and sheep. These proteinsare extremely hydrophobic, have the potential to bind strongly to theprion proteins and to convert them into the β-conformation. They alsohave sequence homologies to one region of the prion protein, which hasbeen shown by others to be involved in the interaction of the so-called‘protein X’ to the sheep prion protein. In addition to having thepossibility of penetrating membranes, BSAS and SCRAPAS have structuralsimilarities to powerful DNA-binding proteins and might autoactivatetheir own expression. They have about 60% overall similarity with eachother. More than 20% of the amino acids are tryptophan as compared tothe average tryptophan content <1% for all proteins for which the aminoacid sequence is known.

Two epitopes (BSAS epi and SCRAPAS epi, 14 amino acids each) wereselected, chemically synthesised and used to produce polyclonalantibodies against BSAS (BSAS pcAb) and SCRAPAS (SCRAPAS pcAb) inrabbits. Two sub-epitopes from within BSAS epi (BSAS mepi) and SCRAPASepi (SCRAPAS mepi), were synthesised, amidated at the c-terminal end andused to affinity purify a specific population of antibodies from BSASpcAb and SCRAPAS pcAb, respectively. These antibodies, and the mepiepitopes, were used in the ELISA tests which are described below.

ELISA Tests

Two types of ELISAs have been developed. One detects in serum theantigens BSAS respectively SCRAPAS, the other detects endogenousantibodies (specific IgG) against BSAS and SCRAPAS mepi epitopes. Thespecificity of all epitopes was confirmed both in commercial scaleisolation of specific populations of IgG's from BSAS pcAb and SCRAPASpcAb, and in the isolation of endogenous IgG from serum samples ofselected BSE-infected cattle and scrapie-infected sheep. Potentialcross-reacting antigenic epitopes were not found in the proteindatabases presently available.

Treatment of Serum Samples for Testing:

BSAS and SCRAPAS are extremely sensitive to freezing. Freezing overnightat −20° C. reduces the reaction of a positive sample by 70-80%; freezingat −80 ° C. eliminates the reaction completely. We believe that thishigh sensitivity to freezing is due to the high tryptophan content ofthe proteins, and the secondary structure enforced on the proteins bythe tandem arrangements of several tryptophan triads in the proteins.The effect of freezing, although not as great for the Ab trap ELISA asfor the Ag trap ELISA, seriously affects both ELISAs (antigen moleculeswith altered confirmation bind irreversible to endogenous IgG inpositive sera which sometimes cause erratic ELISA results in the Ab trapassay). Therefore, frozen samples are not suitable for use in theELISAs. Serum samples must be prepared from freshly drawn blood andstored at 4° C. for such investigations.

Outline of the Tests

The Antigen Trap Test

The wells of micro titre plates were coated with 100 μl of carbonatebuffer, pH 9.6, containing 5 μg/ml mepi epitope-specific antibody, at 4°C. over night. The plates were washed 3× with ELISA washing buffercontaining 0.05% Tween-20. Serum samples, 5 μl, were added to 45 μl ofELISA washing buffer containing 0.075% Tween-20, and incubated at 37° C.for 30 minutes. Wells were washed 5× with the 0.075% Tween-20washing-buffer. 50 μl of second antibody (affinity purifiedSCRAPAS/BSAS-specific antibody, labelled with horseradish peroxidase(HRP)) diluted 1/40 in washing buffer containing 0.05% Tween-20, wasadded to each well and the plates were incubated at 37° C. for 45minutes. The plates were again washed 5× with washing buffer containing0.075% Tween-20. 50 μl substrate solution (Sigma OPD tablet set) wasadded to each well; colour development was allowed for 30 minutes in thedark, and the plates were read at 492 nm.

The Antibody Trap Test

The wells of micro titre plates were coated with 100 μl of carbonatebuffer, pH 9.6, containing 10 μg/ml of each, BSAS mepi and SCRAPAS mepiepitopes, at 4° C. over night. The plates were washed 3× with ELISAwashing buffer containing 0.05% Tween-20. Serum samples, 1.5μl, wereadded to 48.5μl of ELISA washing buffer containing 0.075% Tween-20, andincubated at 37° C. for 30 minutes. Wells were washed 5× with the 0.075% Tween-20 washing buffer. 50 μl of second antibody (mixture ofanti-bovine IgG and anti-sheep IgG antibodies (γ-chain specific,labelled with horseradish peroxidase (HRP)) diluted 1/12000 in washingbuffer containing 0.05% Tween-20, was added to each well and the plateswere incubated at 37° C. for 45 minutes. The plates were again washed 5×with washing buffer containing 0.075% Tween-20. 50 μl substrate solution(Sigma OPD tablet set) was added to each well; colour development wasallowed for 30 minutes in the dark, and the plates were read at 492 nm.

Description of DGDPS Procedure.

In general, first we identified a gene closely related to a gene alreadygenetically or otherwise linked to a certain disease, then isolated themRNA transcribed from the gene from disease tissue or patient's blood,then synthesised cDNA from the isolated mRNA with reverse transcriptase,then amplified the novel cDNA with specific primers which flanked theentire coding region of the cDNA, then we identified the cDNA from thesize following electrophoresis on agarose gel, and finally isolated theunique cDNA from the agarose gel. This allowed us to select out thedesired molecule, if it was expressed, without having to probe severalmillion cDNA clones.

The procedure as used in the present invention and the results obtainedare described in the following examples.

EXAMPLE 1 Discovery of Cattle PrP Prionin BSAS

-   (1) We examined the sequenced regions within the bovine prion    protein gene locus and selected potential complete orf's, i.e. with    acceptable translation initiation sequences (see Kozak, M. Nucleic    Acid Res. 12:857-872 (1984)) and translation termination stop codons    (TAA, TAG or TGA) in place,-   (2) then, orf s fulfilling the above two characteristics were    translated into putative protein sequences using the universal code,-   (3) then we analysed the putative protein with our proprietary    computer assisted protein finger printing technology and obtained    information about the potential biochemical characteristics of the    deduced proteins,-   (4) next the biochemical characteristics of the deduced proteins    were correlated with the known biochemical characteristics of BSE.    Then we determined if the protein was expressed: in order to do this    two potential epitopes were identified/selected within the amino    acid sequence at n-terminal and c-terminal of the deduced protein    using the method of Hopp and Woods, K. R. Proc. Natl. Acad. Sci. USA    78:3824-3828 (1981). The sequences were compared to sequences in    databases and those which appeared to have no homologue within the    databases were selected. Mono-specific polyclonal rabbit antibodies    were prepared against these and purified by immunoaffinity    chromatography on CNBr-activated sepharose 4B (Pharmacia) according    to a procedure which combined the first section of the    recommendation of the manufacturer and the second section as    described in Current Protocols in Molecular Biology (Vol 1)    Ausubel, F. M. et al (ed) John Wiley & Sons NY. N.Y. 1991) and    polyclonal IgG was coupled to horse radish peroxidase. An    enzyme-based immunoassay format “sandwich ELISA” (described in    “ELISA and other Solid Phase Immunoassays” (Kemeny, D. M. et al.    (eds) John Wiley & sons, NY., N.Y. (1988), was used to probe serum,    blood, saliva from BSE positive cows for BSAS using anti-BSAS    IgG-HRP in a colorimetric reaction with “Sigma OPD” as substrate.    The procedure is outlined in section above.

EXAMPLE 2

Detection of BSAS, SCRAPAS and CJAS in Clinical and Other Samples

Prionin proteins were detected using the ELISAs described above. Sincesome populations of the polyclonal anti-TSE antibodies cross react withdomains of the major epitopes of each TSE species, with varying degreesof sensitivity, anti-SCRAPAS-HRP is used as the second antibody fordetecting both BSAS and SCRAPAS, whereas either anti-SCRAPAS HRP oranti-CJAS-HRP is used as the second antibody for detecting CJAS.

EXAMPLE 3

Using another approach, which is not suitable for use as a routine testmethod (with BSAS as the example): BSAS protein was isolated from ˜100ul of serum, from an infected cow on a anti-BSAS antibodyimmuno-affinity column. The isolated protein was subjected to SDS gelelectrophoresis as described by Schägger, H. and von Jabow, G. I. Anal.Biochem. 166:368-379 (1987) or by non-SDS PAGE. Followingelectrophoresis the proteins were subjected to Western blotting orspotted onto nylon membrane and treated with the affinity purifiedantibody. Interaction of the antibody with the protein bound to themembrane was visualised with a chemiluminescent kit purchased fromBio-Rad Inc. according to the manufacturer's instructions (also seeBlake M. S. et al.Anal biochem. 136:175-179 (1984)). MOPAS and HAMPASwere discovered using the same procedure; however, the expression ofthese two proteins in animals with experimental TSE disease has not beeninvestigated so far.

EXAMPLE 4

We have demonstrated, in the case of BSAS and SCRAPAS, a specificassociation (100%) of the proteins with animals confirmed with thedisease or exposed in any manner to the disease; whereas the proteinswere not associated with animals, which have never been exposed to thedisease. BSAS and SCRAPAS was detected in serum of all animalsclinically positive for the diseases and in the majority of animal thathad no demonstrated clinical symptoms of the disease which came fromherds that had even a single case of the disease. CJAS was detected inserum of two CJD victims in one of which was positive for both CJD andAlzheimer's disease.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The success of the present invention derived from our ability to findalternative genes encoding potentially pathogenic proteins BSAS, SCRAPASand CJAS within the PrP genes of cattle, sheep and humans. The discoveryof equivalent genes in mice and hamsters contributed to formulating ourmodel for TSE.

DETAILS OF THE INVENTION

In detail the invention provides the following:

(i) Five protein molecules substantially free of natural contaminants,that encode a protein selected from BSAS, SCRAPAS, CJAS and MOPAS andHAMPAS. In particular the invention provides the aforementioned proteinmolecules wherein the sequence is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4 and SEQ ID NO: 5.

(ii) A method for detecting latent CJD, BSE and scrapie in humans, cowsand sheep respectively by using the ELISA method described above todetect endogenous IgG directed against an epitope in each protein BSAS(SEQ ID NO: 1); SCRAPAS (SEQ ID NO: 2) and CJAS (SEQ ID NO: 3) in bloodof animals and humans respectively, that show no symptoms of thedisease.

(iii) a method for detecting cross infection of animals by animals andhuman by animals by method described in (ii) above to detect thespecific anti-prionin antibody in the blood of a subject.

(iii) A method of detecting contamination of meat, meat products andblood products by, BSAS (SEQ ID NO: 1), SCRAPAS (SEQ ID NO: 2) and CJAS(SEQ ID NO: 3), using one or both the ELISA methods described above todetect either the prionin or traces of a species specific anti prioninIgG in the product tested.

THE MOLECULES OF THE INVENTION

BSAS, SCRAPAS and CJAS (“prionins”) are encoded and expressed fromwithin bovine, sheep and human prion genes:

BSAS “SEQ ID NO: 1”, is a 9.6 Kd basic protein containing 19.75%tryptophan (Trp) residues, a stretch of 22 amino acids (aa) which is apredicted positive DNA regulatory unit and which classifies the proteinas a positive DNA regulator protein. The secondary structure of BSAS isorganised as five (possibly seven) tandem, adjoining, β sheets eachseparated by a β turn. BSAS contains two regions of significant homologyto regions of bovine prion protein between amino acids 90-150. Thelatter region has been suggested by others to be involved in theetiology of BSE in cows. It also has intriguing homology to growthhormone releasing hormone receptor.

SCRAPAS “SEQ ID NO: 2”, is a 7.63 Kd basic protein. It contains 21.87%Trp residues and the identical predicted positive DNA regulatory domain,which is contained in BSAS. The secondary structure of SCRAPAS isorganised as four or five equally spaced β sheets in a pattern almostidentical to BSAS. It has no significant region of homology to the majorsheep prion protein, but has moderate homology to the region (aa 90-150)described above in the bovine prion protein.

CJAS “SEQ ID NO: 3”, is a 7.7 Kd basic protein. It contains 21.53% Trpresidues. CJAS has a Pro-rich N-terminal region and a sequence of 27amino acid, which contains a potential transmembrane helix, whichclassifies it as a transmembrane protein. The secondary structure ofCJAS, which is similar to that of BSAS and SCRAPAS, is organised as fouradjoining β pleats each separated by a β turn. The protein has a domainwith significant homology to a domain in the malaria parasite merozoitemembrane protein which is believed to promote vacuole formation whichgives a spongiform appearance to infected erythrocytes (see Dluzewski A.R. et al J. cell Sci. 92:691-699 (1989)

MOPAS “SEQ ID NO: 4”, is a 5.2 Kd neutral protein. It contains a singletryptophan residue (<0.5%). The secondary structure is organized asthree equally separated β sheets separated by two broad alpha helicalregions. It is not a β sheet structured protein.

HAMPAS “SEQ ID NO: 5”, is a 4.79 Kd basic protein. It contains 26%tryptophan residues. It is basically a CJAS protein truncated at the Nterminal. It shares an antigenic epitope with CJAS. The secondarystructure is organised as three adjoining β pleats, which are arrangedin a similar way to β-sheet 3,4 and 5 of CJAS.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

FIG. 1 a (SEQ ID NO: 1) is the amino acid sequence of BSAS

FIG. 1 b (SEQ ID NO: 2) is the amino acid sequence of SCRAPAS

FIG. 1 c (SEQ ID NO: 3) is the amino acid sequence of CJAS

FIG. 1 d (SEQ ID NO: 4) is the amino sequence of MOPAS

FIG. 1 e (SEQ ID;NO 5) is the amino acid sequence of HAMPAS

FIG. 2 a-c shows the sequence of antigenic epitopes in BSAS; SCRAPAS andCJAS respectively (SEQ ID NOs: 6-8)

FIG. 2 d SCRAPAS epitope appears in BSAS (13 of 14 amino acid residuesin BSAS and 9 of 14 residues in CJAS (SEQ ID NO: 9);

FIG. 2 e part of BSAS epitope appears in SCRAPAS and CJAS (SEQ ID NO:10)

FIG. 3 a ELISA detection of endogenous anti-BSAS IgG in serum of BSEpositive and negative cattle

FIG. 3 b ELISA detection of endogenous anti-BSAS IgG in serum of BSEpositive, BSE negative and clinically negative cows from herds which hadcases of BSE

FIG. 3 c ELISA detection of endogenous anti-SCRAPAS IgG in serum fromscrapie positive and scrapie negative sheep.

FIG. 3 d ELISA detection of endogenous anti-SCRAPAS IgG in serum ofscrapie positive, scrapie negative sheep and of clinically negativesheep that were exposed to scrapie

FIG. 3 e ELISA detection of CJAS protein in serum of patients with CJDand patients with other neurological diseases

FIG. 3 f ELISA detection of anti-CJAS endogenous IgG in serum of CJDpatients and patients with a variety of other neurological diseases.

FIG. 4 sequences of the DNA binding domains of BSAS and SCRAPAS (SEQ IDNO: 11 and 12), (the sequences are 100% homologous) and the sequence ofthe membrane spanning alpha helix of CJAS (SEQ ID NO. 13)

FIG. 5 a. evolutionary structural relationship between BSAS, SCRAPAS,CJAS, HAMPAS and MOPAS suggested from cluster analysis of prions andprionins.

FIG. 5 b the β-sheet structured characteristics of prionins.

Table 1

Antigen trap ELISA test results from blood and urine of cows and sheepwith BSE and scrapie.

BSE dia=clinical diagnosis of animals

cv=EC country herd

neg=no visible clinical symptoms of BSE but exposed to herds withBSE/scrapie

pos=clinical symptoms of BSE/scrapie

nz=test material obtained directly Scrapie/BSE free country

can 43-83=plasma from “40” Canadian cows which were never exposed toBSE*=all samples tested negative in the ELISA.

Table 2.1 & 2.2

Antibody trap ELISA test results from serum obtain from scrapie positivesheep and serum obtained from scrapie negative sheep from a flock neverexposed to scrapie.

Table 3

Antibody trap ELISA test results from serum obtained from BSE positivecows and from BSE negative cows that were never exposed to BSE.

Legend to Figures

FIG. #1 a-e

The amino acid sequence of the prionins, BSAS, SCRAPAS, CJAS MOPAS andHAMPAS deduced from the mRNA sequence.

FIG. 2

Sequence of antigenic epitopes used to prepare polyclonal antibodies.epitopes were chemically synthesized using solid state technology,purified by HPLC and coupled to key hole limpet haemocyanin. IgG waspurified from immune sera and affinity purified on columns of epitopecoupled to CN-sepharose.

FIG. 3

(a) Detection of endogenous anti-BSAS IgG in BSE positive cows but notin well-characterised BSE negative cows (plates coated with BSAS andbound antibody detected with anti bovine IgG HRP.

(b) Detection of endogenous anti-BSAS IgG in serum taken from, cows withclinical BSE, clinically normal cows from a herd with cases of BSE andclinically negative cows not exposed to BSE.

(c) Detection of endogenous anti-SCRAPAS IgG in serum taken from scrapiepositive sheep but not in serum taken from sheep that were never exposedto scrapie (plates coated with BSAS and anti-SCRAPAS was detected withanti-sheep IgG HRP).

(d) Detection of endogenous anti-SCRAPAS IgG in serum from sheep withclinical scrapie, serum from clinically normal sheep from a flock withseveral cases of scrapie and serum from sheep never exposed to scrapie.

(e) Detection of CJAS in serum taken from two CJD victims but not in anormal human or in patients with other neurological conditions (plateswere coated with anti-SCRAPAS IgG and CJAS was detected with anti-CJASIgG HRP, (1=physiological aging, 2=diffuse Lewy bodies, 3=Parkinsons,4=CJD, 5=CJD and Alzheimer's disease, 6=Epilepsy, 7=mixed type dementia,8=AD.

(f) Detection of endogenous anti-CJAS IgG in serum from two CJD patientsbut not in serum from 33 clinically normal humans and humans with avariety of neurodegenerative conditions (CJAS epitope was bound to theelisa wells and bound endogenous anti-CJAS IgG was detected with antihuman Fc specific IgG HRP) 2 & 34=CJD, 20=Alzheimer's disease 1, 3-19,21-33 & 35=normals and other diseases.

g) The presence of BSAS and BSAS complexed to IgG fragments in serumisolated from BSE positive cow. 50 ml of was electrophoresed on cationicnon SDS PAGE and blotted unto nylon membrane the blots were treated withanti BSAS IgG and the presence of BSAS IgG complexed to BSAS wasdetected with a mouse anti rabbit IgG coupled to a chemiluminescentsubstrate, following exposure to X-ray film. IgG was identified in theband with the complex in a separate experiment not shown.

FIG. 4

a, sequence of DNA positive regulators of BSAS and b, SCRAPAS and c, themembrane spanning helix of CJAS.

FIG. 5

a) Evolutionary relationship between the prionins, BSAS, SCRAPAS andCJAS MOPAS and HAMPAS; between bovine PrP, sheep PrP, human PrP, mousePrP and hamster PrP.

b) The β sheet propensity of the prionins.

Physiological Role of BSAS, SCRAPAS and CJAS in the Neuropathology ofBSE, SCRAPIE and CJS.

BSAS, SCRAPAS and CJAS (“prionins”) are expressed from within therespective prion genes (as has been shown for the expression of frameshift proteins from within the human APP gene in Alzheimer's disease),structurally, they are closely related; however, the relationship doesnot appear to be as close as the relationship between the PrPs (seefigure) which are not closely related to the prionins (FIG. 5 a).Prionins are entirely β-sheet derived structures (FIG. 5 b). In thisrespect they resemble snake and scorpion toxins and some insectdefinsins (pore forming proteins) which are predominantly β-sheetstructures (Bontems, F. et al., Science 254:1521-1523 (1991)), butdiffer from these proteins because they lack cysteine residues; howeverprionins contain ˜20 Trp residues which suggest that they are soluble incell membranes and other cellular lipid containing structures.

Furthermore, in prionins β-sheets are separated at regular intervals byβ turns, and, except for very short sequences at c-terminal andn-terminal ends, the proteins are totally hydrophobic molecules; thesestructural characteristics are similar to functional domains of anotherfamily of proteins which interacts with and alters the secondarystructure of other proteins (see PZD and PTB domains Zhou H, et alNature Struct. Biol. 3:388-393 (1996)).

Structural characteristics mentioned above which indicate that prioninsresemble families of pathogenic membrane seeking proteins suggest thatprionins are pathogenic proteins. This is supported by the diseasespecific expression of BSAS, SCRAPAS and CJAS.

Pore forming proteins destroy cells by boring holes in the membranes(Peitsch. M. et al., Mol. Immunol. 27 589-602 (1990)). In the cellcytoplasm they usually aggregate to become soluble. Although neitherBSAS nor SCRAPAS contain a “computer defined” transmembrane signal,because they are, highly hydrophobic molecules, they might mimic poreforming proteins by entering plasma membranes using a hydrophobic wedge(see Hill, H. P. et al., Science 251:1481-1485 (1991)) made up mainly ofTrp residues). Furthermore extremely high concentration of tryptophanresidues in prionins indicate that they may be lipid soluble.

Current dogma holds that PrPs become infectious PrPscs when thesecondary structure of the PrP “flips” from predominantly alpha helix toβ pleats, and this molecular transformation causes the prions to leavethe neuronal membrane and accumulate in extra neuronal spaces (Darcel,C. Vet.Res. Commun. 19:231-252 (1995)). As indicated above, the prioninproteins, can potentially bind to other proteins and enforce the β-plateconfiguration on the proteins they bind to. Like pore boring proteinsprionins can also use the interacting protein as a chaperon fortransport in the soluble form to membrane surfaces. PrPs are expressedin lymphoreticular system and are transported to the brain. Like prions,prionins are found in all TSE diseases. Furthermore, the evolutionaryrelationship to prions (expressed from the same DNA sequences) and thepropensity for binding between different proteins which are expressedfrom the same DNA sequences (see also Baranyi L. et al., Nature Medicine1:894-901 (1995)), suggest that prionins may be specific molecule thatconverts PrPC to PrPSC.

Expression of Prionin Proteins.

As stated earlier, certain observations suggest that BSE is caused whencows are inadvertently fed the remains of scrapie infected sheep, andthat BSE is transmitted to humans who eat infected meat and thisinfection produces a disease phenotype which closely resembles CJS. Thecurrent invention appears to support this hypothesis, because thesethree diseases are associated with three species specific uniqueproteins, which potentially can interact with any other PrP, andtherefore, can “infect” any cell they might enter.

BSAS SEQ ID: 1, SCRAPAS SEQ ID NO: 2 and CJAS SEQ ID NO: 3 are expressedspecifically in cows infected with BSE, sheep infected with scrapie andhumans affected with CJS; all three orf's open from identicaltranslation initiation sequences. BSAS and SCRAPAS are positive DNAbinding proteins that can activate a number of genes and can infectacross species. The latter two proteins are expressed from alternatereading frames within the major prion genes. Hence if cows ingestSCRAPAS in fodder prepared from sheep with scrapie, SCRAPAS mightspuriously bind to a PrP in the lymphoid cells located in the stomach,get transported to the brain, activate translation of BSAS whichconverts more prions to the pathogenic form and hence initiate BSE inthese animals. BSAS entering humans by way of the food chain mightactivate translation (or transcription) of CJAS, which has the sametranslation initiation sequence as BSAS and SCRAPAS, and initiate thesymptoms of CJS.

From the above discussions it should be obvious that prionins can bypassthe blood brain barrier. Once in the brain they might interact withneuronal membrane binding sites usually occupied by PrP. Furthermoresince prionins are lipid soluble molecules they can penetrate the plasmamembrane, diffuse through lipid layers and enter neurons.

Using BSAS as the example, we believe that this might work as follows:

The PrP protein in neuronal plasma membrane is a target either specificor opportunistic for prionins. BSAS endogenously expressed in, orentering a cell by an external route, interacts with PrPs. On binding tothe PrP the rigid, β-sheet structure of BSAS forces the flexibleconformation of the PrP to change in a manner which accommodates theBSAS β-sheet structure. This results in the formation of a prion (seeHarrison. S. C., Cell 86:343-344 (1996). The soluble BSAS-Prion complextraverses the cytoplasm and leaves the cell through the membrane, entersthe lymph vesicular transport system in which it transverses the bloodbrain barrier and arrives at the surface of a specific subpopulation ofneurons. The latter are the usual targets of PrPs. On contact with aneuron BSAS releases from the prion and penetrates the neuronalmembrane. The prion cannot enter the membrane in the converted state andis left in the intraneuronal spaces where aggregation occurs with otherdiscarded prions. Alternatively, on release from BSAS the PrP flips backinto the original alpha helical PrP state. The cycle of events isamplified as BSAS that enters the neuron overactivates the expression ofPrPs.

Since BSAS and SCRAPAS contain the identical positive DNA regulatorysequence FIG. 4, they are likely to activate similar genes and probablyautoactivate their own transcription or translation.

Alternatively, prionins can be expressed from a promoter system located3′ downstream, which is believed to program expression of PrP. WhereasPrPs are expressed in appropriate cells throughout life and appear to benormal essential components of neurons, all be it, with functionspresently unknown, (but see Schmerling et al., Cell 93:203-214 (1998)).Prionins are probably expressed, secreted and targeted (specificreceptors or opportunistic) to neuronal and other non-neuronal cellsonly in diseased animals.

Support for the notion that prionins are pathogenic was provided by thefinding that prionins are bound to a variety of immunoglobulin moleculesin the blood of all animals that are clinically positive for BSE/scrapieand humans with clinical CJD. The antibody was also found in asignificant number of clinically negative animals that were exposed toBSE or scrapie. The endogenous antibody was never found in animals thatwere not exposed to BSE/scrapie nor in clinically normal humans thatwere not exposed to CJD. This indicates that the subject's immune systemconsidered prionins foreign and tried to neutralize them when they wereexpressed in animals and humans. The immune response to prionins may beextremely important in the control of TSE diseases especially incontrolling cross infections. It is also important in detecting latencyand of course in presymptomatic diagnosis of TSE diseases. Furthermoreit appears likely that prionin expression can be initiated when theappropriate cells, are exposed to some environmental toxin (e.g.,microbial or viral infection, intracellular metabolic toxic by productsor stress). Such infections might have the effect of first stressing thesubject's immune system in a way that it is unable to cope efficientlywith other task and secondly the infecting agent may also activate theexpression of prionins. This renders the immune system less effectiveand permits prionins to escape and enter cells where they interact withPrPs.

In summary, prionins acting as pore forming proteins or acting whilebound to the prion in the neurons disrupts the lipid layer and enterneurons where they activate expression of a number of genes includingPrP and prionins. Prionin molecules remaining on or within the membraneelicit a response from the neuronal immune system. This immune responsetargets cells containing the prionin molecules and hence selectivelydestroys these prionin-bearing cells, which takes on the character of anautoimmune reaction against endogenous neurons. Because this autoimmuneaction selectively targets cells bearing prionins on the surfacesurrounding cells are for the most part left untouched and the result isthe vacoulated spongiform appearance of TSE brains.

The Uses of the Molecules of the Present Invention

A. Diagnostic Uses

Since BSAS, SEQ ID NO: 1; SCRAPAS SEQ ID NO: 2 and CJAS SEQ ID NO: 3,are not expressed at a detectable level by normal animals or humans, thedetection of these molecules in a tissue or fluid sample (such as abiopsy sample, or of blood or urine or saliva) is indicative of thepresence of the disease in that subject even before any clinicalsymptoms are present. In Example # 6, the results of 103, mixed, blindedsamples including blood, serum and urine were tested using the ELISAtest outlined in FIG. 3 a. All samples from clinically positive samplestested positive for BSAS or SCRAPAS, all clinically normal animals thatwere never exposed to BSE or scrapie tested negative for BSAS or SCRAPASwhereas up to 30% of the clinically negative animals that were exposedto BSE tested positive. The latter animals were presymptomatic for thedisease.

We have used a sensitive sandwich ELISA approaches for detecting BSAS,SCRAPAS and CJAS in cow, sheep and human material and for detectinglatency and cross contaminations in animals, foods and blood products;however, the detection of these molecules may be done by any of avariety of immunological methods; a large number of suitable immunoassayformats have been described (Yolken, R. H., Rev. Infect. Dis. 4:35(1982); Collins, W. P., In: Alternative Immunoassays, John Wiley & Sons,NY (1985); Ngo, T. T. et al., In: Enzyme Mediated Immunoassay, PlenumPress, NY (1985); incorporated by reference herein.

Example: The antibody prepared against the epitope in hamster prioninHAMPAS, SEQ ID NO: 5, can be used to detect CJAS. In lieu of suchantibodies, equivalent binding molecules, such as antibody fragments(F(ab′), F(ab′)2, single chain antibodies, etc.), recombinantantibodies, chimeric antibodies, etc. may be employed.

As indicated above, immunoassay formats may employ labelled antibodiesto facilitate detection. Radioisotopic immunoassays (“RIAs”) have theadvantages of simplicity, sensitivity, and ease of use. Radioactivelabels are of relatively small atomic dimension, and do not normallyaffect reaction kinetics. Such assays suffer, however, from thedisadvantages that, due to radioisotopic decay, the reagents have ashort shelf life, require special handling and disposal, and entail theuse of complex and expensive analytical equipment. RIAs are described inLaboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S., et al., North Holland Publishing Company, NY (1978), with particularreference to the chapter entitled “An Introduction to Radioimmune Assayand Related Techniques” by Chard, T., incorporated by reference herein.

No single enzyme is ideal for use as a label in every conceivableimmunometric assay. Instead, one must determine which enzyme is suitablefor a particular assay system. Criteria important for the choice ofenzymes are turnover number of the pure enzyme (the number of substratemolecules converted to product per enzyme site per unit of time), purityof the enzyme preparation, sensitivity of detection of its product, easeand speed of detection of the enzyme reaction, absence of interferingfactors or of enzyme-like activity in the test fluid, stability of theenzyme and its conjugate, availability and cost of the enzyme and itsconjugate, and the like. Examples of suitable enzymes, which can beused, include peroxidase, acetylcholine esterase, alpha-glycerolphosphate dehydrogenase, alkaline phosphatase, asparaginase,b-galactosidase, catalase, among many others. Peroxidase and urease areamong the more preferred enzyme labels, particularly because ofchromogenic pH indicators, which make its activity readily visible tothe naked eye.

B. Therapeutic Uses

Significantly, the present invention provides means for treating BSE,scrapie and CJS. Such treatment may be either “prophylactic” or“therapeutic.” A prophylactic treatment is one that is provided inadvance of any clinical symptom of BSE, scrapie or CJS in order toprevent or attenuate any subsequent onset of the disease. A therapeutictreatment is one that is provided in response to the onset of a symptomof BSE, scrapie or CJS and serves to attenuate an actual symptom of thedisease.

In one embodiment, such treatment is provided by administering to ananimal or human in need of such treatment an effective amount of anantibody, or an antibody fragment (F(ab′), F(ab′)2, single chainantibodies, etc.) or a combination of the above that is capable ofbinding to BSAS, SCRAPAS or CJAS. As used herein, an effective amount isan amount sufficient to mediate a clinically significant change in theseverity of a symptom, or a clinically significant delay in the onset ofa symptom.

As will be appreciated, for acute administration, monospecificpolyclonal or monoclonal antibodies (or fragments of either) may beadministered. More preferably, and especially for chronicadministration, the use of non-immunogenic antibodies is preferred. Suchmolecules can be pseudo-homologous (i.e. produced by any species, butaltered to a form that is immunologically indistinct from humanantibodies). Examples of such pseudo-homologous molecules include“humanized” (i.e. non-immunogenic in a human) prepared by recombinant orother technology. Such antibodies are the equivalents of the monoclonaland polyclonal antibodies, but are less immunogenic, and are bettertolerated by the patient.

Humanized anti CJAS can be produced, for example by replacing animmunogenic portion of each antibody with a corresponding, butnon-immunogenic portion (i.e. chimeric antibodies) (Robinson, R. R. etal., International Patent Publication PCT/US86/02269; Akira, K. et al.,European Patent Application 184,187; Taniguchi, M., European PatentApplication 171,496; Morrison, S. L. et al., European Patent Application173,494; Neuberger, M. S. et al., PCT Application WO 86/01533; Cabilly,S. et al., European Patent Application 125,023; Better, M. et al.,Science 240:1041-1043 (1988); Liu, A. Y. et al., Proc. Natl. Acad. Sci.USA 84:3439-3443 (1987); Liu, A. Y. et al., J. Inmunol. 139:3521-3526(1987); Sun, L. K. et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987);Nishimura, Y. et al., Canc. Res. 47:999-1005 (1987); Wood, C. R. et al.,Nature 314:446-449 (1985)); Shaw et al., J. Natl.Cancer Inst.80:1553-1559 (1988); all of which references are incorporated herein byreference). General reviews of “humanized” chimeric antibodies areprovided by Morrison, S. L. (Science, 229:1202-1207 (1985)) and by Oi,V. T. et al., BioTechniques 4:214 (1986); which references areincorporated herein by reference. Suitable “humanized” antibodies canalternatively be produced by CDR or CEA substitution (Jones, P. T. etal., Nature 321:552-525 (1986); Verhoeyan et al., Science 239:1534(1988); Beidler, C. B. et al., J. Immunol. 141:4053-4060 (1988); all ofwhich references are incorporated herein by reference).

C. Administration of the Molecules of the Present Invention

Additional pharmaceutical methods may be employed to control theduration of action. Control release preparations may be achieved throughthe use of polymers to complex or absorb the agents. The controlleddelivery may be exercised by selecting appropriate macromolecules (forexample polyesters, polyamino acids, polyvinyl pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine, sulphate) and the concentration of macromolecules as well asthe methods of incorporation in order to control release.

Having now generally described the invention, through references andexamples that makes it more readily understood by any one sufficientlyskilled in the art, it must be pointed out that these are not intendedto be limiting of the present invention, unless specified.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains,and as may be applied to theessential features herein before set forth and as follows in the scopeof the claims.

TABLE 1 sample source tissue BSE dia. scrapie dia. ELISA  1 cv serum negna −  2 cv serum neg na +  3 cv serum pos na +  4 cv serum neg na −  5cv serum pos na +  6 cv serum pos na +  7 cv serum pos na +  8 cv serumneg na +  9 cv serum neg na − 10 cv serum pos na + 11 cv serum pos na +12 cv serum neg na − 13 cv serum pos na + 14 cv serum neg na − 15 cvserum neg na + 16 cv serum neg na − 17 cv serum neg na + 18 cv serum posna + 19 cv serum neg na − 20 cv serum pos na + 21 cv serum neg na + 22cv serum neg na − 23 cv serum neg na − 24 cv serum neg na − 25 cv serumneg na − 26 cv serum pos na + 27 cv serum pos na + 28 cv serum pos na +29 cv serum pos na + 30 cv serum pos na + 31 cv urine pos na + 32 cvurine pos na + 33 cv urine pos na + 34 cv urine pos na + 35 cv urine posna + 36 cv urine neg na + 37 cv urine neg na + 38 nz urine neg na − 39nz urine neg na − 40 nz urine neg na − 41 nz urine neg na − 42 nz urineneg na − 43-83 can plasma all neg na   − * 84 cv plasma na neg − 85 cvplasma na neg − 86 cv plasma na neg − 87 cv plasma na pos + 88 cv plasmana neg − 89 cv plasma na pos + 90 cv plasma na neg − 91 cv plasma napos + 92 cv plasma na pos + 93 cv plasma na neg − 94 cv plasma na neg −95 cv plasma na pos + 96 cv plasma na pos + 97 cv plasma na pos + 98 cvplasma na neg − 99 cv plasma na neg − 100  cv plasma na pos + 101  cvplasma na neg − 102  cv plasma na pos + 103  cv plasma na pos +

TABLE 2-1 mean absorbance status mean absorbance status 367 POS 211 NEG373 POS 117 NEG 389 POS 182 NEG 449 POS 19 NEG 437 POS 143 NEG 409 POS131 NEG 419 POS 189 NEG 450 POS 189 NEG 362 POS 122 NEG 362 POS 127 NEG360 POS 172 NEG 331 POS 183 NEG 333 POS 204 NEG 419 POS 206 NEG 331 POS206 NEG 392 POS 40 NEG 383 POS 51 NEG 360 POS 73 NEG 368 POS 117 NEG 357POS 152 NEG 391 POS 78 NEG 383 POS 111 NEG 347 POS 128 NEG 378 POS 112NEG 394 POS 138 NEG 387 POS 199 NEG 333 POS 202 NEG 410 POS 147 NEG 379POS 122 NEG 453 POS 137 NEG 413 POS 80 NEG 431 POS 16 NEG 414 POS 108NEG 376 POS 139 NEG 412 POS 181 NEG 366 POS 184 NEG 348 POS 10 NEG 463POS 181 NEG 350 POS 134 NEG 362 POS 213 NEG 416 POS 83 NEG 417 POS 115NEG 334 POS 164 NEG 380 POS 123 NEG 380 POS 217 NEG 346 POS 177 NEG 418POS 103 NEG 373 POS 208 NEG 349 POS 65 NEG

TABLE 2-2 399 POS 115 NEG 401 POS 167 NEG 331 POS 129 NEG 448 POS 48 NEG341 POS 123 NEG 473 POS 179 NEG 397 POS 223 NEG 426 POS 90 NEG 352 POS165 NEG 368 POS 167 NEG 433 POS 116 NEG 342 POS 5 NEG 598 POS 58 NEG 334POS 104 NEG 397 POS 158 NEG 358 POS 129 NEG 538 POS 120 NEG 512 POS 58NEG 336 POS 118 NEG 389 POS 153 NEG 349 POS 179 NEG 433 POS 188 NEG 375POS 146 NEG 92 NEG 119 NEG 123 NEG 208 NEG 184 NEG 105 NEG

TABLE 3 Mean absorbance Status 669 POS 455 POS 684 POS 518 POS 572 POS546 POS 453 POS 474 POS 668 POS 457 POS 588 POS 572 POS 813 POS 579 POS496 POS 515 POS 589 POS 60 NEG 14 NEG 44 NEG 22 NEG 206 NEG 22 NEG 53NEG 214 NEG 31 NEG 22 NEG 196 NEG 98 NEG 188 NEG 259 NEG 39 NEG 179 NEG96 NEG 169 NEG 133 NEG 204 NEG 89 NEG 152 NEG 41 NEG 102 NEG 30 NEG 111NEG 103 NEG 105 NEG 290 NEG 172 NEG 131 NEG 147 NEG 58 NEG 118 NEG 33NEG 41 NEG 268 NEG 153 NEG 137 NEG 101 NEG 121 NEG 83 NEG 74 NEG 66 NEG78 NEG 200 NEG 191 NEG 89 NEG 120 NEG 111 NEG 82 NEG 187 NEG 238 NEG 114NEG 41 NEG 110 NEG 70 NEG 239 NEG 41 NEG 118 NEG

1. A protein molecule free of natural contaminants selected from thegroup consisting of BSAS, SCRAPAS, CJAS, and HAMPAS the sequence ofwhich is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:5,respectively.
 2. A purified antibody that cross reacts with either BSAS,SCRAPAS, CJAS or HAMPAS, the sequence of which is SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3 and SEQ ID NO:5, respectively.
 3. The antibody ofclaim 2, wherein the antibody is produced by immunizing appropriateanimals with polypeptide domains of BSAS or SCRAPAS or CJAS or HAMPAS,respectively.
 4. The antibody of claim 2 that cross reacts with eitherBSAS, SCRAPAS or CJAS, wherein said antibody is produced endogenously bya cow or by a sheep or by a human, respectively.
 5. The antibody ofclaim 2, wherein the antibody is an antibody fragment, a single chainantibody, a monospecific polyclonal antibody, a monoclonal antibody, arecombinant antibody or a humanized antibody.
 6. A method for thedetection of a TSE in humans or animals comprising detecting in a tissueor fluid sample a protein as defined by SEQ ID NO:1, SEQ ID NO:2, or SEQID NO:3 or an antibody that cross reacts with any of the proteins asdefined by SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
 7. The method ofclaim 6 wherein the TSE is bovine spongiform encephalopathy (BSE),scrapie disease or Creutzfeldt-Jakob Syndrome (CJS).